Endoplasmic reticulum oxidase 1α is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP–cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1␣ (ERO1␣) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1␣ in HSCs remains unclear. Here, we show that ERO1␣ is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1␣ siRNA or an ERO1␣ shRNA-expressing plasmid, expression of ERO1␣ was completely silenced. Silencing of ERO1␣ expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1␣ expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1␣ expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1␣ plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.
Hepatic stellate cells (HSCs) 2 are located in the space of Disse between the basolateral surfaces of hepatocytes and the antiluminal sides of sinusoidal endothelial cells (1,2). In the liver, under physiological conditions, HSCs mainly accumulate vitamin A as retinyl ester in lipid droplets in the cytoplasm. Upon injury to or inflammation of the liver, HSCs lose vitamin A and transdifferentiate to myofibroblastic cells, so-called activated HSCs, which produce and secrete excessive extracellular matrices, including collagens, suggesting that activated HSCs are a major contributor to liver fibrosis and attractive target cells for anti-fibrotic therapy (3)(4)(5). Indeed, some studies have demonstrated that resolution of collagens in the liver of cirrhotic animal models is triggered by in vivo delivery of siRNA against the collagen-specific chaperone heat shock protein 47 (HSP47) or collagen type 1␣1 to activated HSCs (6,7).
During liver fibrosis, there is dramatic proliferation of activated HSCs, accompanied by excessive secretion and deposition of collagens in liver tissue. Our previous study and other studies also revealed that activated HSCs proliferate through the interaction of integrins with collagen molecules secreted by the HSCs (8 -10). Collagens secreted by activated HSCs are cleaved by membrane type 1-matrix metalloproteinase (MT1-MMP) on the cell surface, leading to exposure of the RGD motif on the surfaces of collagen molecules. Integrin ␣v␤1 on activated HSCs binds to the RGD motif in collagen molecules, resulting in promotion of proliferation and survival of activated HSCs. Therefore, the collagen/MT1-MMP/integrin signaling axis is indispensable for the proliferation and survival of activated HSCs; however, the molecular mechanism by which the collagen/MT1-MMP/integrin signaling axis in HSCs is regulated remains elusive.
Intramolecular disulfide bond formation of the membrane and secreted proteins mainly relies on the protein disulfide isomerase (PDI) machinery, which accepts electrons from client cysteine thiols (11). Endoplasmic reticulum oxidase 1 (ERO1) has been identified as a major client of the PDI machin-ery (12,13). ERO1 accepts electrons from reduced PDI and hands them over to oxygen molecules, catalyzing oxygen-mediated disulfide bond formation (14,15). In mammals, two types of ERO1 (ERO1␣ and ERO1␤) have been identified, and loss-of-function mutations in mammalian ERO1 genes result in rather subtle phenotypes (16,17). Mice lacking ERO1␣, which is ubiquitously expressed in several tissues and cells, have an abnormal cardiac response to adrenergic stimulation, and mice lacking ERO1␤, which is specifically expressed in pancreatic ␤ cells, develop mild nonprogressive pancreatic ␤ cell dysfunction with glucose intolerance (18). The combined loss of function of ERO1␣ and ERO1␤ has been shown to compromise the extracellular matrix in mice and interfere with the intracellular maturation of procollagen (19). Collagen type 1 consists of two molecules of collagen type 1␣1 and one molecule of collagen type 1␣2 and also has some disulfide bonds between these molecules, leading to the formation of a triple helix structure (20). It has been reported that the disulfide bond in collagen molecules is formed by PDI (21,22), suggesting that ERO1␣ plays a crucial role in the disulfide bond formation of collagen molecules, which is involved in the maintenance of HSC proliferation. However, the functional significance of ERO1␣ in HSCs remains unclear. Here we investigated the expression of ERO1␣ in human HSCs and examined the effect of ERO1␣ silencing on the proliferation of HSCs via the collagen/MT1-MMP/integrin signaling axis to explore the functional significance of ERO1␣ in HSCs.

Silencing of ERO1␣ inhibits proliferation of HSCs
We first confirmed the expression of ERO1␣ in human hepatic stellate cell lines (LX-2 and LI90) and primary human HSCs. Quantitative RT-PCR and Western blotting showed that ERO1␣ is expressed in LX-2 cells, LI90 cells, and primary HSCs (Fig. 1, A and B). ERO1␣ was co-localized with HSP47, which is a collagen-specific chaperone and a marker of the endoplasmic reticulum (ER), in LX-2 cells (82.2%), LI90 cells (83.1%), and primary HSCs (80.6%) (Fig. 1C), indicating that ERO1␣ is expressed and mainly localizes in the ER in HSCs.
To clarify the significance of ERO1␣ in HSCs, we investigated the proliferation of HSCs by silencing ERO1␣ expression. When LX-2 cells were transfected with three batches of ERO1␣ siRNA, the expression of ERO1␣ mRNA and protein was completely silenced ( Fig. 2A). Because the knockdown efficiency of ERO1␣ expression in LX-2 cells transfected with ERO1␣-A siRNA was the highest among the three batches of ERO1␣ siRNAs, and transfection with ERO1␣-A siRNA inhibited the expression of ERO1␣ protein in LX-2 cells, LI90, cells and primary HSCs (Fig. 2B), we used ERO1␣-A siRNA for the cell proliferation assay. Silencing of ERO1␣ expression induced impaired proliferation of LX-2 cells, LI90 cells, and primary HSCs but did not induce cell death during a period of 5 days after transfection with ERO1␣-A siRNA (Fig. 2, C and D).
Based on the results of the ERO1␣ siRNA-silencing experiment, we generated HSCs stably expressing ERO1␣ shRNA by transfection of two cell lines (LX-2 and LI90) with ERO1␣ shRNA-expressing plasmids to establish HSCs constitutively silencing ERO1␣ expression (Fig. 3A). Proliferation of the cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA was markedly suppressed compared with that of the cell lines stably expressing control shRNA (Fig. 3B). It has been reported that HSCs proliferate through activation of the FAK/MAPK signaling pathway and Akt/IB signaling pathway (23)(24)(25). Thus, we and primary HSCs was examined by quantitative RT-PCR. n.s., not significant. B, immunoblots of ERO1␣ protein in LX-2 cells, LI90 cells, and primary HSCs. C, immunofluorescent staining of ERO1␣ and HSP47 in LX-2 cells, LI90 cells, and primary HSCs. HSP47 served as an endoplasmic reticulum marker. The degree of co-localization of ERO1 with HSP47 in cells was determined by NIH ImageJ. Scale bars ϭ 100 m.

The role of ERO1␣ in collagen and MT1-MMP biosynthesis
examined the phosphorylation of FAK and activation of the Raf-1/MEK/MAPK signaling pathway in two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA. As shown in Fig.  3C, the phosphorylation of FAK and activation of Raf-1/MEK/ MAPK in both LX-2 cells and LI90 cells were markedly inhibited by silencing of ERO1␣ expression. Intriguingly, phosphorylation of Akt at Ser-473 and Thr-308 and phosphorylation of IB at Ser-32/36 were clearly detected in two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA. Activation of the Akt/IB signaling pathway in HSCs sustains their survival apart from the induction of cell proliferation (10,23,24), suggesting that activation of the Akt/IB signaling pathway is responsible for the survival of HSCs stably expressing ERO1␣ shRNA regardless of impaired cell proliferation via inactivation of the FAK/Raf-1/MEK/MAPK signaling pathway and that ERO1␣ plays a critical role in the proliferation of HSCs.

ERO1␣ regulates the secretion of collagen type 1 from HSCs
We examined the secretion of collagen type 1 from HSCs stably expressing ERO1␣ shRNA. ELISA and immunoblotting of collagen type 1 showed that the amount of collagen type 1 secreted from the two hepatic stellate cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA was significantly smaller than that secreted from the two hepatic stellate cell lines (LX-2 and  LI90) or from two hepatic stellate cell lines (LX-2 and LI90) stably expressing control shRNA (Fig. 4A). On the other hand, the expression levels of collagen 1␣1 and 1␣2 mRNAs in the two hepatic stellate cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA were slightly increased compared with those in the counterparts (Fig. 4B). Immunoblotting and immunofluorescence revealed that collagen type 1 had accumulated excessively in the two hepatic stellate cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA compared with the amount in two cell lines (LX-2 and LI90) stably expressing control shRNA (Fig.  4, C and D). Furthermore, immunoblotting under non-reducing conditions showed that collagen type 1 formed a triple helix in LX-2 cells and the LX-2 stably expressing control shRNA, but only a single chain of collagen type 1 was detected in LX-2 cells stably expressing ERO1␣ shRNA (Fig. 4E). These results indicated that silencing of ERO1␣ expression in HSCs causes impaired disulfide bond formation of collagen type 1, resulting in inhibition of collagen type 1 secretion.

The role of ERO1␣ in collagen and MT1-MMP biosynthesis
We then investigated whether the impaired proliferation of HSCs by silencing of ERO1␣ expression is rescued by addition of collagen type 1. Surprisingly, proliferation of two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA progressed only modestly with addition of collagen type 1, and the rate of proliferation was significantly lower than the rates of proliferation of two cell lines (LX-2 and LI90) stably expressing control shRNA (Fig. 5, A and B). These results suggest that an additional obstacle other than impaired secretion of collagen type 1 is involved in the inhibited proliferation of HSCs by silencing of ERO1␣ expression.

Silencing of ERO1␣ inhibits autophagy in HSCs via activation of Akt
Some studies have demonstrated that unfolded or immature procollagens that have accumulated in the ER in fibroblasts and HSCs are mainly degraded by autophagy (26 -28). Thus, we examined autophagy in two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA. Immunofluorescence and immunoblotting of LC3 showed that there was no aggregation of LC3 or an increased level of LC3II in the two cell lines with or without ERO1␣ expression (Fig. 6, A and B). Formation of autophagosomes was also not observed in LX-2 cells stably expressing control shRNA or ERO1␣ shRNA (Fig. 6C). Intriguingly, phosphorylation of adenosine monophosphate-activated protein kinase ␣1 (AMPK␣1), an inducer of autophagy, was detected in

The role of ERO1␣ in collagen and MT1-MMP biosynthesis
two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA but not in two cell lines (LX-2 and LI90) stably expressing control shRNA (Fig. 6B). On the other hand, phosphorylation of ULK1 at Thr-555, a target site of AMPK␣1, was inhibited in two cell lines (LX-2 and LI90) by silencing ERO1␣ expression (Fig.  6D). Because silencing of ERO1␣ expression in HSCs stimulated phosphorylation of Akt (Figs. 3C and Fig. 6B), we further examined the phosphorylation status of mammalian target of rapamycin (mTOR), an inhibitor of ULK1. As expected, phosphorylation of mTOR at Ser-2448, a target site of Akt, was observed in two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA (Fig. 6B). Treatment with an Akt inhibitor at a concentration of 5 or 10 M induced phosphorylation of ULK1 at Thr-555 and the formation of autophagosomes in LX-2 cells stably expressing ERO1␣ shRNA (Fig. 6, D and E). Treatment with the Akt inhibitor partially inhibited the expression of collagen 1␣1 mRNA and collagen 1␣2 mRNA in two cell lines (LX-2 and LI90) stably expressing control shRNA and ERO1␣ shRNA compared with that in the counterparts (Fig. 6F). However, although there was no significant difference in the expression levels of collagen 1␣1 mRNA and collagen 1␣2 mRNA between cells stably expressing control shRNA with the Akt inhibitor and cells stably expressing ERO1␣ shRNA with the Akt inhibitor, immunoblotting revealed that excessive accumulation of intracellular collagen type 1 in cells (LX-2 and LI90) stably expressing ERO1␣ shRNA was markedly ameliorated by treatment with the Akt inhibitor (Fig. 6G). These results indicate that silencing of ERO1␣ inhibits autophagy through activation of the Akt signaling pathway, causing excessive accumulation of collagens in HSCs.

MT1-MMP is regulated by ERO1␣ in HSCs
It has been shown that the proliferation of HSCs progresses through the modification of collagens with MMP2 and MT1-MMP, which are expressed by HSCs themselves (8,10). We examined the expression of MMP2 and MT1-MMP in HSCs stably expressing control shRNA or ERO1␣ shRNA. The expression levels of MT1-MMP mRNA, but not those of MMP2 mRNA, in two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA were slightly higher than those in two cell lines (LX-2 and LI90) expressing control shRNA (Fig. 7A). On the other hand, the levels of MT1-MMP protein in two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA were significantly lower than those in the counterparts, reflecting the
Intracellular proteins and misfolded proteins are degraded by the ubiquitin-proteasome pathway and by autophagy (31,32). In this study, phosphorylation of Akt, but not of AMPK␣1, was detected, and aggregation of LC3 was not observed in two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA (Figs. 3C and Fig. 6), suggesting that the ubiquitin-proteasome pathway is responsible for the decrease in the level of MT1-MMP protein induced by silencing of ERO1␣ expression. Thus, we examined whether recovery of MT1-MMP proteins in HSCs stably expressing ERO1␣ shRNA is induced by treatment with a proteasome inhibitor, MG132. As shown in Fig. 7C, the expression of MT1-MMP protein in two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA was completely rescued by treatment with 5 M MG132. On the other hand, flow cytometric analysis showed that MT1-MMP was not localized in the cellular membrane but in the cytoplasm in LX-2 cells stably expressing ERO1␣ shRNA, regardless of recovery of MT1-MMP protein expression by treatment with MG132 (Fig.  7D). MT1-MMP protein has disulfide bond formation in the hemopexin domain (33), suggesting that unfolded MT1-MMP protein that accumulates in HSCs by silencing of ERO1␣ expression is degraded through the ubiquitinproteasome pathway.

MT1-MMP-cleaved collagens promote proliferation of ERO1␣-silenced HSCs
As described above, secretion of collagens was impaired and degradation of MT1-MMP was enhanced in HSCs by silencing of ERO1␣ expression. The proliferation of HSCs is enhanced through the interaction of integrin ␤1 with collagens (10). Because the expression of integrin ␤1 in HSCs was sustained despite the silencing of ERO1␣ expression (Fig. 3A), we examined whether addition of MT1-MMP-cleaved collagens promotes the proliferation of HSCs stably expressing ERO1␣ shRNA. A cell proliferation assay showed that the proliferation of two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA was significantly enhanced by addition of MT1-MMPcleaved collagen type 1, as was also observed in two cell lines (LX-2 and LI90) stably expressing control shRNA (Fig. 8, A  and B).

Discussion
Excessive deposition of extracellular matrix containing collagens in response to injury or inflammation is a leading cause of liver fibrosis, which mainly depends on activation of HSCs, which synthesize collagen and promote their own proliferation via interaction with collagen. Thus, an understanding of the mechanism by which HSCs regulate the biosynthesis of collagen will provide a meaningful clue for practical anti-fibrotic therapy of organs. In this study, we demonstrate for the first time that ERO1␣, an oxidative enzyme of PDI that forms intramolecular disulfide bonds of membrane and secreted proteins  (14,15), is expressed in human HSCs and that silencing of ERO1␣ expression markedly inhibits the proliferation of HSCs, accompanied by inactivation of the integrin/FAK/Raf/MEK/ ERK signaling pathway. Silencing of ERO1␣ expression in HSCs resulted in excessive intracellular accumulation and impaired secretion of collagen type 1. Western blot analysis under non-reducing conditions showed that silencing of ERO1␣ expression causes accumulation of single chains of collagen type 1 in HSCs, indicating inhibition of disulfide bond formation between collagen molecules. The proliferation of HSCs is mainly dependent on the interaction of integrins with collagens secreted from HSCs (8,10,26,34). Collagen type 1 consists of two molecules of collagen type 1␣1 and one molecule of collagen type 1␣2 and also has some disulfide bonds between these molecules, leading to the formation of a triple helix structure (20). It has been reported that the combined loss of function of ERO1␣ and ERO1␤ interferes with the intracellular maturation of pro-collagen (19). Therefore, ERO1␣ may play a critical role in the proliferation of HSCs through the proper structural formation and secretion of collagens.

The role of ERO1␣ in collagen and MT1-MMP biosynthesis
It has been reported that unfolded or immature procollagens that have accumulated in fibroblasts and HSCs are degraded by an autophagy-dependent degradation system to prevent them from exposure to prolonged ER stress, which leads to apoptosis (26 -28). Activation of autophagy, which consists of the formation of autophagosomes and activation of the AMPK/ULK1 signaling pathway, is indispensable for the maintenance of cell survival under serum starvation or low-glucose conditions (35)(36)(37). Moreover, inhibition of autophagy evokes an accumulation of misfolded or aggregated proteins in the ER (38 -40). This study shows excessive accumulation of unfolded collagen type 1 in human HSCs by silencing ERO1␣ expression. Aggregation of Figure 6. Silencing of ERO1␣ in human hepatic stellate cells inhibits autophagy through activation of Akt. A, immunofluorescence of LC3B in LX-2 cells stably expressing control shRNA or ERO1␣ shRNA. LX2 cells cultured in serum-starved medium were used as a positive control. Scale bars ϭ 50 m. B, immunoblots of phospho-AMPK␣1, AMPK␣1, phospho-Akt, Akt, phospho-mTOR, mTOR, phospho-ULK1, ULK1, and LC3B in two cell lines (LX-2 and LI90) stably expressing control shRNA or ERO1␣ shRNA. C, formation of autophagosomes in LX-2 cells stably expressing control shRNA or ERO1␣ shRNA. Scale bars ϭ 2 m. D, immunoblots of ULK1 in two cell lines (LX-2 and LI90) stably expressing control shRNA or ERO1␣ shRNA after treatment with an Akt inhibitor (5 and 10 M). E, formation of autophagosomes in LX-2 cells stably expressing control shRNA or ERO1␣ shRNA after treatment with an Akt inhibitor (10 M). Scale bars ϭ 2 m. F, expression of collagen type 1␣1 and collagen type 1␣2 mRNAs in two cell lines (LX-2 and LI90) stably expressing control shRNA or ERO1␣ shRNA after treatment with an Akt inhibitor (10 M). *, p Ͻ 0.05; n.s., not significant. G, immunoblots of collagen type 1 protein in two cell lines (LX-2 and LI90) stably expressing control shRNA or ERO1␣ shRNA after treatment with an Akt inhibitor (10 M). LC3, formation of autophagosomes, and phosphorylation of ULK1 were not triggered, although phosphorylation of AMPK was induced, by silencing of ERO1␣ expression in human HSCs. Intriguingly, silencing of ERO1␣ expression in human HSCs induced activation of the Akt/mTOR signaling pathway (Fig. 3B), which suppresses autophagy through inhibition of phosphorylation of ULK1 (41,42). We found that treatment of ERO1␣-silenced human HSCs with an Akt inhibitor ameliorated excessive accumulation of collagen type 1 and induced activation of autophagy, accompanied by formation of autophagosomes and phosphorylation of ULK1. Although the molecular mechanism by which activation of Akt is stimulated by silencing of ERO1␣ expression remains unclear, the results imply that silencing of ERO1␣ expression triggers inhibition of autophagy via activation of Akt, which is responsible for excessive accumulation of collagens in HSCs.

The role of ERO1␣ in collagen and MT1-MMP biosynthesis
It has been shown that proliferation of HSCs progresses through modification of collagens with MMP2 and MT1-MMP, which are expressed by HSCs themselves (8, 10). MT1-MMP has disulfide bond formation in the hemopexin domain in its molecule (33), suggesting that expression and synthesis of MT1-MMP are involved in the impaired proliferation of human HSCs by silencing ERO1␣ expression. In this study, silencing of ERO1␣ expression resulted in progressive degradation of MT1-MMP protein through the proteasome pathway, although no reduction of MT1-MMP mRNA expression was detected. Flow cytometric analysis also revealed that localization of MT1-MMP on the surface of human HSCs was inhibited by silencing ERO1␣ expression. These results suggest that ERO1␣ may regulate the trafficking of MT1-MMP to the cell surface through proper structural formation of MT1-MMP protein. Collagens secreted by activated HSCs are cleaved by MT1-MMP on the cell surface, leading to exposure of the RGD motif to the surfaces of collagen molecules (10). Integrin ␣v␤1 on activated HSCs binds to the RGD motif in collagen molecules, resulting in promotion of proliferation and survival of activated HSCs. This study shows that the proliferation of ERO1␣-silenced human HSCs is completely recovered by

The role of ERO1␣ in collagen and MT1-MMP biosynthesis
treatment with MT1-MMP-cleaved collagen type 1. Treatment with MT1-MMP-cleaved collagen type 1 also triggered activation of the FAK/Raf/MEK/ERK signaling pathway in ERO1␣-silenced human HSCs. These results imply that ERO1␣ plays a critical role in collagen secretion and MT1-MMP biosynthesis in human HSCs.
Numerous studies on the resolution of organ fibrosis and cirrhosis have been carried out. Excessive secretion of collagens from activated stellate cells and myofibroblasts is a leading cause of organ fibrosis and cirrhosis, suggesting that elimination of these causative agents is necessary for anti-fibrotic therapy (43)(44)(45). In fact, inhibition of collagen secretion from stellate cells has been demonstrated to partially induce the resolution of organ fibrosis (6, 46 -49). Suppression of proliferation and reduction of the number of activated stellate cells are also critical for fibrosis regression. For example, suppression of HSC proliferation through p53 activation has been reported to contribute to the reversion of fibrosis (50). It has also been shown that inhibition of expression of NFB and decreased expression of the tissue inhibitor of metalloproteinase 1 reduce the number of activated HSCs, resulting in regression of liver fibrosis (51). However, targeting only one component in a complex signaling pathway may not be sufficiently potent, and targeting more than one molecule is considered to be a meaningful clue for anti-fibrotic therapy. In this context, this study showed that silencing of ERO1␣ expression results in impaired secretion of collagens, activation of AMPK, and progressive degradation of MT1-MMP protein, causing suppression of the proliferation of human HSCs. Therefore, targeted disruption of ERO1␣ in stellate cells may be one of the therapeutic modalities for tissue fibrosis.
In conclusion, this study demonstrates that ERO1␣ is expressed and localizes in the ER of human HSCs and that silencing of ERO1␣ expression inhibits the proliferation of HSCs through impaired secretion of collagen type 1, inhibition of autophagy, and progressive degradation of MT1-MMP protein. Treatment with MT1-MMP-cleaved collagen type 1 restored the inhibited proliferation of HSCs by silencing of ERO1␣ expression through activation of the collagen/MT1-MMP/integrin ␤1/focal adhesion kinase signaling axis. These results suggest that ERO1␣ plays a crucial role in the proliferation of HSCs through regulation of collagen secretion and biosynthesis of MT1-MMP protein.

Culture of hepatic stellate cells
The human hepatic stellate cell lines LX-2 and LI90 were purchased from Millipore (Billerica, MA) and from the JCRB

The role of ERO1␣ in collagen and MT1-MMP biosynthesis
Cell Bank (National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan), respectively. Primary human HSCs were obtained from ScienCell Research Laboratories (Carlsbad, CA). LX-2 cells, LI90 cells, and primary human HSCs were cultured in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Gibco) and 100 M ascorbic acid (Sigma).

Immunofluorescence
Cells were fixed with 4% paraformaldehyde (Wako Pure Chemicals, Tokyo, Japan). After washing with PBS (Life Technologies), the cells were permeabilized with PBS supplemented with or without 0.5% Triton X-100 for 3 min at room temperature and treated with 5% goat serum in PBS for 60 min at room temperature. The cells were then incubated with primary antibodies against ERO1␣ (Millipore), HSP47 (Abcam, Cambridge, UK), collagen type 1 (Abcam), and MT1-MMP (Abcam). After washing with PBS, the cells were incubated with the following secondary antibodies: Alexa 488 -conjugated goat anti-rabbit IgG (Invitrogen) and Alexa 555-conjugated goat anti-mouse IgG (Invitrogen). Finally, the cells were counterstained with DAPI and analyzed with an immunofluorescence microscope. The degree of co-localization of ERO1␣ with HSP47 in the cells was determined by National Institutes of Health ImageJ.

Cell proliferation assay
LX-2 cells, LI90 cells, and primary HSCs were plated at 2 ϫ 10 4 cells/well in a 6-well plate and cultured for 24 h. After transfection with control siRNA or ERO1␣-A siRNA at a concentration of 10 nM, the treated cells were cultured for 120 h. Proliferation of LX-2 cells treated with control siRNA or ERO1␣-A siRNA was determined by trypan blue staining. Cell proliferation assays were carried out 24, 48, 72, 96, and 120 h after transfection with siRNA.

Establishment of HSCs stably expressing ERO1␣ short hairpin RNA
LX-2 cells and LI90 cells were transfected with plasmids containing a control shRNA sequence (OriGene) or ERO1␣ shRNA sequence (5Ј-AGAGCATTCTACAGACTTATA-TCTGGCCT-3Ј). 48 h after transfection with the plasmid, the treated cells were cultured in DMEM supplemented with 10% FBS and 2 g/ml puromycin (Invitrogen) for 2 weeks to select cell clones stably expressing control shRNA or ERO1␣ shRNA.

The role of ERO1␣ in collagen and MT1-MMP biosynthesis
To confirm silencing of ERO1␣ expression in the selected cell clones, the expression of ERO1␣ mRNA and protein was determined by quantitative RT-PCR and Western blotting.

Enzyme-linked immunosorbent assay
Cells plated at 2 ϫ 10 4 cells/well in 6-well plates were cultured for 96 h. All samples were stored at Ϫ80°C until use. The amount of mature collagen type I in supernatants was determined using a human collagen type 1 ELISA kit (ACEL). Absorbance was determined at 450 nm. The amount of secreted collagen type I was determined by normalizing the amount of collagen type 1 in supernatants with cell number.

Transmission electron microscopy
LX-2 cells and LX-2 cells stably expressing control shRNA or ERO1␣ shRNA cultured with or without Akt inhibitor (Akt inhibitor IV, Millipore) were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3). After washing with 0.1 M cacodylate buffer (pH 7.3), the cells were post-fixed in 1% OsO 4 and 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer. Samples were stained with uranyl acetate for 2 h at room temperature and then washed, dehydrated, and embedded in Epon 812. Ultrathin sections were cut with a diamond knife and stained with lead citrate. Samples were examined with an electron microscope (JEM-3200FS, JEOL) at an acceleration voltage of 300 kV.

Flow cytometric analysis
LX-2 cells stably expressing control shRNA or ERO1␣ shRNA were cultured in DMEM supplemented with 10% FBS, and 100 M ascorbic acid, and with or without 5 M proteasome inhibitor MG132 for 12 h and further cultured in DMEM supplemented with 10% FBS for 24 h. The treated cells were incubated with isotype control rabbit IgG (Abcam) and a primary antibody against MT1-MMP at 4°C for 30 min. After washing with PBS, the cells were incubated with Alexa 488 -conjugated goat anti-rabbit IgG as a secondary antibody. Fluorescence of the treated cells was determined by flow cytometry (FACS Canto, BD Biosciences).

Statistical analysis
All experiments were carried out independently three times. Results are shown as means Ϯ S.D. Comparisons between two groups were performed using Student's t test, with p Ͻ 0.05 considered to be statistically significant.
Author contributions-M. F. and A. Yoneda designed all experiments and performed most of them. A. Yoneda wrote the manuscript. K. S. S. and M. K. performed Western blotting and the cell growth assay. A. Yoneda, N. T., K. M., A. Yokoyama, and Y. T. prepared the manuscript.